In article <DBJxqL.Azr at murdoch.acc.virginia.edu>,
William D. Woolfolk <wdw7m at virginia.edu> wrote:
>Yes, you can use a similar method. I have frequently amplified directly
>from bacteria by pulling up a small amount of bacterial material with a
>pipet tip, and adding it to a 50ul PCR reaction with the Taq omitted. I
>heat to 95C for 15 minutes,then add Taq and cycle as I would for normal
>DNA template-based PCR. The only problem I ever had was using a massive
>clump of bacteria, which decreased amplification some. A small swipe is
>more than adequate for PCR, so do not overdo it. Good luck.
A 10 ul reaction works fine, and (in my hands) one doesn't need to
heat-treat for 15 min (but I do use a 2 min 95 deg first step after
adding Taq). Just touching a micropipette tip to the colony (you
don't need to see the cells you've removed) and mixing it in with the
master mix also suffices to give nice bands.
john
--
John Nash Institute for Biological Sciences *
Email: nash at nrcbsa.bio.nrc.ca National Research Council of Canada * .*
World Wide Web: http://cansnd.cisti.nrc.ca/~nash/home.html *
Disclaimer: Opinions are mine, not NRC's